Lymph transport occurs against a hydrostatic pressure gradient and thus relies critically on the intrinsic contractile function of lymphatic muscle, the lymphatic pump. Failure of this pump system is associated with many types of lymphedema. Little is known about why lymphatic vessels become dysfunctional, but clinical studies reveal an elevated lymphatic diastolic pressure, enlarged diameter, impaired or absent contractions, and what appear to be incompetent valves. Collectively these findings point to underlying problems with the lymphatic pacemaker, pump and valves. In this proposal we will test the mechanisms leading to contraction and valve dysfunction at pressures experienced by the lymph pump during the development of lymphedema. Recently we developed methods to measure the membrane potential of lymphatic smooth muscle (LSM) in isolated, pressurized mouse lymphatics and patch clamp methods to study selected ion currents in LSM cells. We will apply those methods to transgenic mouse models to study the ionic basis of the electrical pacemaker that drives spontaneous and pressure-regulated contractions. Our preliminary results challenge the existing dogma about the pacemaking mechanism in LSM and suggest it is controlled by interactions between voltage- gated Ca2+ channels and KCNQ K+ channels. Our new data show the same channels operate in human LSM. We have developed methods to test valve and pump function in isolated single lymphangions from the mouse when the vessel is subjected to increasing output pressure, simulating the load on a vessel in a dependent extremity. Our results provide new insights into how the lymph pump fails when pressure is elevated. The pump either gradually weakens until it cannot eject, or the output valve locks open catastrophically. Importantly, we find that both types of pump failure can be corrected by the administration of norepinephrine (NE) to activate the pacemaker while preserving contractile strength and tone for normal valve gating. Although pump failure occurs in some normal vessels subjected to elevated pressure, susceptibility to failure is increased of mice deficient in the transcription factor FOXC2, which controls the development and maintenance of lymphatic valves. Foxc2+/- mice recapitulate the human disease lymphedema distichiasis. Our central hypothesis is that lymphatic pacemaking, pump strength, and valve function are closely interrelated such that failure of any one disables the lymph pump and leads to lymphedema. We propose that pump dysfunction can be rescued by pharmacological intervention in normal, Foxc2+/- and inducible Foxc2-/- mice. This hypothesis will be tested by 3 aims: 1) Determine the ionic basis of the lymphatic action potential and pacemaking. 2) Determine the ionic mechanisms by which pressure modulates the lymphatic pacemaker. 3) Determine the mechanisms by which NE modulates the lymphatic pacemaker and can rescue a lymphatic vessel that is made dysfunctional by an elevated pressure load or valve defect. This approach to treating a failed lymph pump represents a potential new strategy for treating a key underlying cause of lymphedema.